Zhou, Shanshan (2016) Antibiotic biosynthesis and its transcriptional regulation in Streptomyces bacteria. PhD thesis, University of Warwick.

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Abstract

Streptomyces, the largest genus of Actinobacteria, are renowned for their ability to produce a wide variety of specialised metabolites, including many clinically used antibiotics and other bioactive natural products. Biosynthesis of these specialised metabolites is often tightly regulated by transcriptional regulators, some of which are responsive to signalling molecules, e.g., 2-alkyl-4-hydroxymethylfuran-3-carboxylic acids (AHFCAs), thus initiating a series of regulatory events.

The biosynthetic role of MmfL in AHFCA assembly has been investigated in vitro using a synthetic substrate. MmfL has been shown to catalyse an AfsA-like reaction, involving condensation of an ACP-bound β-ketothioester and dihydroxyacetone phosphate to form a phosphorylated butenolide intermediate.

The interactions between DNA, AHFCA ligands and ArpA-like repressors, MmfR and SgnR, have been investigated in vitro using electrophoretic mobility shift assays. This work leads to a better understanding of the regulation mechanism in specialised metabolite biosynthesis.

Heterologous expression of a putative pyochelin-like gene cluster, sven0503-sven0517 from S. venezuelae ATCC 10712, has previously been shown to result in the production of thiazostatin and watasemycin as the main metabolic products of the cluster. Isopyochelin, a structural isomer of pyochelin, was also identified and characterised by comparison with synthetic standards. Sven0516 has previously been identified as the thiazoline reductase required for the biosynthesis of all of the metabolic products of the cluster. The class B radical SAM methylase Sven0515 was shown to be responsible for the methylation of thiazostatin to give watasemycin. This is the first experimentally validated example of such a reaction in the biosynthesis of a nonribosomal peptide. From incorporation experiments using stereospecifically deuterium-labelled cysteine, it has been demonstrated that Sven0515-mediated methylation proceeds with abstraction of the pro-R hydrogen atom and results in inversion of stereochemistry at the methylating position. The absolute stereochemistry of thiazostatin and watasemycin has been reassigned on the basis of the absolute configuration determined for isopyochelin.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QD Chemistry
Library of Congress Subject Headings (LCSH):	Streptomyces -- Genetics, Actinobacteria, Metabolites -- Synthesis, Biosynthesis, Genetic transcription -- Regulation
Official Date:	September 2016
Dates:	Date Event September 2016 Submitted
Institution:	University of Warwick
Theses Department:	Department of Chemistry
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Challis, Gregory L.
Format of File:	pdf
Extent:	xxiii, 201 leaves : illustrations, charts
Language:	eng

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